Activation of the Yeast Hippo Pathway by
Phosphorylation-Dependent Assembly of Signaling
Complexes
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Citation
Rock, J. M., D. Lim, L. Stach, R. W. Ogrodowicz, J. M. Keck, M.
H. Jones, C. C. L. Wong, et al. “Activation of the Yeast Hippo
Pathway by Phosphorylation-Dependent Assembly of Signaling
Complexes.” Science 340, no. 6134 (May 17, 2013): 871–875.
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http://dx.doi.org/10.1126/science.1235822
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American Association for the Advancement of Science (AAAS)
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Published in final edited form as:
Science. 2013 May 17; 340(6134): . doi:10.1126/science.1235822.
Activation of the Yeast Hippo Pathway by PhosphorylationDependent Assembly of Signaling Complexes
Jeremy M. Rock1,2, Daniel Lim1, Lasse Stach3, Roksana W. Ogrodowicz3, Jamie M. Keck4,*,
Michele H. Jones4, Catherine C. L. Wong5, John R. Yates III5, Mark Winey4, Stephen J.
Smerdon3, Michael B. Yaffe1, and Angelika Amon1,2,†
1David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology,
Cambridge, MA 02139, USA
2Howard
Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA
02139, USA
3Division
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of Molecular Structure, MRC National Institute for Medical Research, The Ridgeway,
London NW7 1AA, UK
4Department
of Molecular, Cellular, and Developmental Biology, UCB 347, University of
Colorado, Boulder, CO 80309, USA
5Department
of Chemical Physiology, Scripps Research Institute, La Jolla, CA 92037, USA
Abstract
Scaffold-assisted signaling cascades guide cellular decision-making. In budding yeast, one such
signal transduction pathway called the mitotic exit network (MEN) governs the transition from
mitosis to the G1 phase of the cell cycle. The MEN is conserved and in metazoans is known as the
Hippo tumor-suppressor pathway. We found that signaling through the MEN kinase cascade was
mediated by an unusual two-step process. The MEN kinase Cdc15 first phosphorylated the
scaffold Nud1. This created a phospho-docking site on Nud1, to which the effector kinase
complex Dbf2-Mob1 bound through a phosphoserine-threonine binding domain, in order to be
activated by Cdc15. This mechanism of pathway activation has implications for signal
transmission through other kinase cascades and might represent a general principle in scaffoldassisted signaling.
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Signaling complex assembly must be tightly controlled in both space and time. The use of
protein scaffolds and phosphorylation-dependent protein-protein interactions is central to
this spatiotemporal control. The mitotic exit network (MEN) is a conserved guanosine
triphosphatase (GTPase) signaling cascade that governs exit from mitosis (1, 2). During
anaphase, MEN signaling complexes assemble at spindle pole bodies (SPBs) (3, 4) to trigger
exit from mitosis and to couple this cell cycle transition with nuclear position (5). The Raslike GTPase Tem1 and the Polo protein kinase Cdc5 coordinately recruit the Hippo-like
Copyright 2013 by the American Association for the Advancement of Science; all rights reserved.
†
Corresponding author: angelika@mit.edu.
*Present address: Division of Hematology and Medical Oncology and OHSU Knight Cancer Institute, Oregon Health and Science
University, Portland, OR 97239, USA.
Supplementary Materials
www.sciencemag.org/cgi/content/full/science.1235822/DC1
Materials and Methods
Figs. S1 to S14
Tables S1 to S4
References
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kinase Cdc15 to SPBs (3). Once localized to SPBs, Cdc15 is activated to phosphorylate the
kinase Dbf2 and its coactivator Mob1 (6). Phosphorylation activates Dbf2-Mob1, which
then promotes the release of the MEN effector protein phosphatase Cdc14 from the
nucleolus, resulting in exit from mitosis (2).
Scaffold proteins serve as assembly platforms for kinase cascades and may function as
signaling insulators (7). Our results show that, rather than functioning as a passive platform
onto which MEN components assemble, the SPB-resident MEN scaffold Nud1 is a dynamic
participant in MEN signal transmission. Nud1 is a phospho-protein and its phosphorylation
increases during mitosis (fig. S1, A to C, and table S1) (8–10). We generated a NUD1 allele
in which the 38 high-confidence mitotic phosphorylation sites and 4 lower-confidence sites
were mutated to alanine (henceforth nud1-42A). The nud1-42A protein was stable and
localized to SPBs, but phosphorylation during mitosis was reduced (fig. S1, B to D).
To examine the effects of the nud1-42A allele on MEN activity, we introduced the allele into
a strain expressing the temperature-sensitive nud1-44 allele under the control of the
galactose-inducible and glucose-repressible GAL1-10 promoter. GAL-nud1-44 nud1-42A
cells, like MEN loss-of-function mutants, arrested in late anaphase with inactive Dbf2-Mob1
and nucleolar-restricted Cdc14 under conditions in which nud1-44 is inactive (Fig. 1, A and
B, and fig. S1, E and F). Thus, nud1-42A cells are defective in MEN signaling.
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Localization of the MEN components Tem1, Cdc15, Dbf2, and Mob1 to SPBs is essential
for Dbf2-Mob1 activation and requires NUD1 (3, 4, 11). Localization of Nud1, Bfa1 [a
Tem1 GTPase-activating protein (GAP) complex component], Tem1, and Cdc15 was
normal in nud1-42A cells (12) (fig. S2, A to D), but Mob1 and Dbf2 were absent from SPBs
(fig. S2, E and F). nud1-42A cells also harbored mispositioned anaphase spindles and
detached astral microtubules (fig. S1F). Thus, the nud1-42A allele is defective in recruitment
of Dbf2-Mob1 to SPBs and astral microtubule anchorage (11).
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Further analyses (12) (fig. S3, A to C) revealed that Nud1 T78 was especially critical for
MEN signaling, with two additional residues, S53 and S63, contributing to this function. A
NUD1 allele carrying alanine substitutions of S53, S63, and T78 (nud1-3A; table S1) caused
an anaphase arrest when overexpressed in the presence of wild-type NUD1 (fig. S3C) and
failed to restore viability to cells expressing the GAL-nud1-44 allele grown under restrictive
conditions (fig. S3D). The anaphase delay caused by a NUD1 allele that included the S53A
and S63A mutations but not T78A was minor (fig. S3C). Replacing S53, S63, and T78 with
residues that mimic phosphorylation (Asp or Glu) disrupted Nud1 function (12), precluding
us from examining the consequences of constitutive phosphorylation of these residues. S53,
S63, and T78 are conserved across fungal orthologs (fig. S4). Thus, these residues may have
similarly important roles in other fungal species.
Localization of Mob1 to SPBs was disrupted in nud1-3A cells as it was in nud1-42A cells
(Fig. 1C), but astral microtubule organization was not affected (fig. S5A). In contrast, a
NUD1 allele in which all mitotic phosphorylation sites were mutated to alanine with the
exception of S53, S63, and T78 (nud1-39A allele; table S1) facilitated normal Mob1
localization and restored viability to cells expressing the GAL-nud1-44 allele under
restrictive conditions (Fig. 1, D and E). nud1-39A cells did exhibit a 2-min delay in exit
from mitosis (fig. S5B), suggesting that other phosphorylation sites in Nud1 play a very
minor role in MEN activation. nud1-39A cells also harbored mispositioned anaphase
spindles and detached astral microtubules (fig. S5, A, C, and D). The spindle positioning and
astral microtubule anchoring defect of nud1-39A cells was suppressed by forcing
localization of the astral microtubule anchor Spc72 to SPBs (fig. S5E), suggesting that
nud1-39A cells are defective in Spc72 recruitment. Indeed, Spc72 localization to SPBs was
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impaired in nud1-39A cells (fig. S5F). These results distinguish the two Nud1 functions:
MEN activation and astral microtubule anchorage.
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Mob1 bound to SPBs in the absence of Dbf2 (Fig. 3A), but Dbf2 did not bind to SPBs in the
absence of Mob1 (Fig. 3B). This observation, together with the finding that Mob1 and Nud1
form a complex in a CDC15-dependent manner (Fig. 2A), suggests that Mob1 binds Nud1
in a phosphorylation-dependent manner. To test this, we assayed the interaction of
recombinant Mob1 with Nud1 phosphopeptides in vitro. Glutathione S-transferase (GST)–
Mob1 (amino acids 79 to 314) that contains the conserved Mob1 core but not the N terminus
unique to yeast (14) preferentially bound to phosphorylated T78 Nud1 peptides (Fig. 3C).
Oriented peptide library screening (12) revealed that Mob1 displayed a preference for Y or F
in the −2 position and preferred aliphatic, hydrophobic, or R residues in the +1 to +4
positions. The Nud1 protein sequence surrounding T78 shows features consistent with these
results (fig. S9, A to C). We measured binding of GST-Mob1 to filter-bound peptides
predicted to be optimal ligands on the basis of the peptide library screening. Mob1 bound to
these optimal peptides in a phosphorylation-specific manner (Fig. 3D). The binding of Mob1
to the optimal ligand was stronger than that to the Nud1 pT78 peptide (dissociation constant
Kd of 174 nM and 2.4 μM, respectively; fig. S9, D and E). These values are similar to those
observed for the interaction of FHA phosphopeptide binding domains with their optimal and
physiologic peptides (15, 16). Thus, Mob1 directly binds Nud1 T78 in a phosphorylationdependent manner.
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Next, we sought to identify the kinase(s) that phosphorylates Nud1 S53, S63, and T78.
Cdc15 is a likely candidate because it, like Nud1 phosphorylation, is essential for Dbf2Mob1 SPB localization (fig. S6) (13). Mob1 coimmunoprecipitated with Nud1 in anaphasearrested cells in which CDC15 is active (cdc14-3 cells) but not in cells in which CDC15 is
inactive (cdc14-3 cdc15-2 cells; Fig. 2A). Cdc15 kinase activity was also sufficient for
localization of Dbf2-Mob1 to SPBs. Constitutive targeting of Cdc15 to SPBs by fusing
CDC15 to the SPB (CDC15-SPB) (3) led to recruitment of Dbf2-Mob1 to SPBs soon after
expression of CDC15-SPB and induced premature phosphorylation of Nud1 (Fig. 2B and
fig. S7). To directly test whether Cdc15 phosphorylates Nud1 in vivo, we raised phosphospecific antibodies to S53, S63, and T78 (fig. S8). The antibodies bound to wild-type Nud1
only in anaphase cells (Fig. 2C). T78 phosphorylation depended on CDC15, S63
phosphorylation was partially dependent, and S53 phosphorylation was not (Fig. 2D).
Consistent with this, premature targeting of Cdc15 to SPBs was sufficient to cause
phosphorylation of Nud1 T78 and S63, but not S53 (Fig. 2E). In vitro, Cdc15
phosphorylated a recombinant N-terminal fragment of Nud1 (amino acids 1 to 150) but not
the nud1-3A protein (Fig. 2F). Thus, Cdc15 phosphorylates Nud1 on residues T78 and S63
during anaphase to promote binding of Dbf2-Mob1 to Nud1.
We attempted to crystallize human (hMob1) and yeast Mob1 as complexes with the Nud1
pT78 and optimal phosphopeptides. Of these combinations, hMob1 bound to the peptide
TVARIYHpSVVRYAPS yielded crystals of sufficient quality for structure determination
and refinement at a resolution of 2.1 Å (table S4). The phosphopeptide binds across a
shallow pocket on the Mob1 surface (Fig. 4A and fig. S10A). The structural basis for
selection of aromatic residues in the pSer/pThr −2 position (fig. S9, A and B) is explained
by the packing of the phenolic side chain of Tyr −2 against the aliphatic portion of K84. In
yeast Mob1, this lysine is substituted by a proline, which would also make favorable van der
Waals interactions. Discrimination for small-to medium-sized hydrophobic residues at the
+1 and +2 positions is also explained by their interactions with a largely conserved
hydrophobic depression on the Mob1 surface.
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The structure explains the phospho-dependence of Mob1 binding through extensive
interactions of the phosphoserine with three basic residues (K132, R133, and R136) that
form part of a positively charged pocket located on the face opposite to that of the Dbf2interacting surface. In yeast Mob1, three structurally equivalent arginine residues, R253,
R254, and R257, form the conserved basic pocket and directly coordinate a sulfate ion
derived from the crystallization mother liquor (Fig. 4B and fig. S10B) (14). Thus, the Mob1
protein family contains a distinct class of phosphoserine-threonine binding domains.
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To test whether these arginine residues are important for Mob1 binding to Nud1, we assayed
the interaction of GST-mob1-R253A, R254A, R257A (henceforth mob1-3RA) with
phosphopeptides. The mob1-3RA protein was properly folded (fig. S11A), but failed to
interact with Nud1 pT78 peptides (Fig. 4C). Thus, the basic pocket of Mob1 mediates
phosphopeptide binding. Phosphopeptide binding was also essential for Mob1 function in
vivo. The mob1-R→A proteins interacted with Dbf2 (fig. S11B) but did not localize to
SPBs (Fig. 4D and fig. S11C) nor support viability (Fig. 4E and fig. S11D). Furthermore,
these cells lacked Dbf2 kinase activity and failed to release Cdc14 from the nucleolus or to
exit from mitosis (Fig. 4F). This cell cycle arrest was not suppressed by a dominant,
constitutively active Dbf2 allele (DBF2-HyA; fig. S11E) (17). Thus, even if the defect in
Dbf2 activation is corrected in mob1-3RA cells, the failure of mob1-3RA to bind
phosphopeptides and presumably target Dbf2 to its relevant substrates results in an inability
to exit from mitosis. Indeed, the phosphopeptide binding property of Mob1 may also be
critical for targeting Dbf2-Mob1 to its substrates. Mob1 binds experimentally verified
phosphorylation sites in the Dbf2-Mob1 substrate Hof1 (fig. S12) (18).
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Our results show that MEN signaling requires an unusual two-step process (Fig. 4G). Rather
than directly activating its downstream kinase Dbf2-Mob1, Cdc15 first creates phosphodocking sites on the MEN scaffold Nud1. Nud1 phosphorylation recruits Dbf2-Mob1 to
SPBs followed by Cdc15-dependent activation of Dbf2-Mob1. As Cdc15 alone is sufficient
to activate Dbf2-Mob1 in vitro (6), why do cells use a two-step mechanism for Dbf2-Mob1
activation? A two-step scaffold-assisted mechanism of activating downstream kinases may
afford many advantages. In addition to allowing for greater regulatory power, this
mechanism mitigates the “biphasic effect” in which scaffold proteins can exhibit
concentration-dependent titration effects (19). Increasing concentrations of scaffold proteins
initially favor interaction of partner proteins but, at higher concentrations, will sequester
partner proteins into separate complexes. The dependency of terminal kinase binding on
scaffold phosphorylation may ensure that terminal kinases only engage in complexes in
which their upstream kinases are bound and active. Such a protective mechanism predicts
that MEN signaling ought to be resistant to the overexpression of individual signaling
components, and this is what we observe (fig. S13).
A two-step mechanism may also facilitate signal transmission and effector activation at
distinct sites in the cell. Dbf2-Mob1 activated by Cdc15 at SPBs must phosphorylate Cdc14
in the nucleolus. This predicts that the Mob1-Nud1 interaction must be dynamic. In
agreement, fusion of MOB1 to NUD1 (MOB1-NUD1) resulted in the constitutive
localization of Dbf2 to SPBs (fig. S14, A and B) but, despite the proper localization pattern,
did not restore viability to mob1-77 cells at restrictive conditions (fig. S14C). Indeed,
expression of MOB1-NUD1 was lethal in wild-type cells because Cdc14 could not be
released from the nucleolus (fig. S14, D to F). This lethality appears to result from failure of
Dbf2-Mob1 to dissociate from SPBs because a Mob1-Nud1 fusion in which Mob1 could be
proteolytically released from Nud1 restored proper MEN regulation (Fig. 4, H and I).
Furthermore, lethality caused by expression of MOB1-NUD1 was not suppressed by
expression of a dominant, constitutively active Dbf2-HyA allele (fig. S14G), indicating that
even hyperactive Dbf2 that cannot dissociate from SPBs is not functional. During MEN
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signaling, dynamic interaction between Mob1 and Nud1 may be achieved by Cdc15 first
generating a high-affinity scaffold docking site for Dbf2-Mob1. Phosphorylation of Dbf2Mob1 by Cdc15 could then, in addition to activating the kinase, decrease affinity to the
Nud1 scaffold, thereby promoting the dissociation of Dbf2-Mob1 from SPBs.
In this study, we have uncovered a distinct mechanism of scaffold-assisted activation of a
kinase cascade. We propose that the two-step activation of terminal kinases may be a
common feature of scaffold-assisted signaling that provides a potentially general means to
avoid the biphasic effect and organize signaling pathways in which the site of pathway
activation differs from the essential site of action.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
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We thank I. Cheeseman, F. Solomon, and the Amon lab for comments and E. Schiebel for reagents. Coordinates
and structure factors for the hMob1-phosphopeptide complex have been deposited with the Protein Data Bank
(PDBID: 4JIZ). Supported by the NIH (GM056800 to A.A.; CA112967 and ES015339 to M.B.Y; P41 RR011823
to J.R.Y. III and T. N. Davis, principal investigator; and GM51312 to M.W.); MRC UK (U117584228) to S.J.S;
MRC centenary fellowship to L.S.; NIH F32 GM086038 to J.M.K.; and NSF Predoctoral Fellowship to J.M.R.
A.A. is an investigator of the Howard Hughes Medical Institute.
References and Notes
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Fig. 1. Dbf2-Mob1 recruitment to SPBs and MEN activation requires Nud1 phosphorylation
(A and B) Dbf2 kinase activity and cell cycle progression in GAL-nud1-44 NUD1 (A29878)
and GAL-nud1-44 nud1-42A (A29881) cells. Cells were arrested in G1 with α-factor and
released under conditions in which nud1-44 is inactive (12). (C) Mob1 localization in
anaphase NUD1 (A29453) and nud1-3A (A31169) cells. (D) Mob1 localization in anaphase
NUD1 (A24631) and nud1-39A (A31477) cells. DAPI, 4′,6-diamidino-2-phenylindole; DIC,
differential interference contrast. (E) Growth of 10-fold serial dilutions of A2587, A29248,
A29685, A29500, and A32295 cells on YEP plates containing either galactose and raffinose
(YEPRG) or glucose (YEPD) (12).
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Fig. 2. Phosphorylation of Nud1 by Cdc15 in anaphase promotes Mob1 recruitment to SPBs
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(A) Nud1-3V5 was immunoprecipitated from anaphase-arrested A31602 and A31661 cells,
and bound Mob1 (IPα-HA), Nud1 (IPα-V5), Nud1 phosphorylated at T78 (IPα-pT78), total
Mob1 (input α-HA), and total Nud1 (input α-V5) were examined. (B) Localization of Cdc15
and Mob1 in G1/S, metaphase, and anaphase MET3-CDC15-SPB MOB1-mCherry (A28499)
cells grown in medium lacking methionine to induce expression of CDC15-SPB. (C)
Phosphorylation of Nud1 T78 (α-pT78), S63 (α-pS63), S53 (α-pS53), and total Nud1 during
a synchronized cell cycle (A24513). Percent meta-phase (% meta) and anaphase (% ana)
cells are shown. (D) Phosphorylation of Nud1 as in Fig. 2C in anaphase-arrested NUD1-3V5
cdc14-3 (A29851) and NUD1-3V5 cdc14-3 cdc15-2 (A31661) cells. (E) Phosphorylation of
Nud1 as in Fig. 2C in S-phase–arrested NUD1-3V5 (A24513) and NUD1-3V5 MET3CDC15-SPB (A31422) cells (12). CDC15-SPB expression was induced as in Fig. 2B. (F)
Phosphorylated Nud1, immunoprecipitated Cdc15 [Cdc15 IP; wild type (WT): A24957;
kinase-dead (KD) Cdc15: A30371], and GST fusion protein substrate added to the kinase
reaction (Substrate) are shown.
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Fig. 3. Phosphorylation-dependent binding of Mob1 to Nud1
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(A) Mob1 localization in anaphase MOB1-eGFP (A24631) and MOB1-eGFP dbf2Δ TAB6-1
(A32656) cells. TAB6-1 is a dominant active CDC14 allele that was necessary to keep dbf2Δ
cells alive (20). (B) Dbf2 localization in anaphase DBF2-eGFP (A29921) and DBF2-eGFP
GAL-mob1-77 (A32654) cells. eGFP, enhanced green fluorescent protein. (C) Bead-bound
phosphorylated (pT78, pS53, or pS63) or nonphosphorylated (T78, S53, or S63) Nud1
peptides were incubated with GST-Mob1 and input and eluates analyzed. (D) Binding of
GST-Mob1 to an array of peptide spots. Abbreviations for the amino acid residues are as
follows: A, Ala; H, His; I, Ile; R, Arg; S, Ser; T, Thr; V, Val; and Y, Tyr.
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Fig. 4. Mob1 binding to phospho-Nud1 through a conserved basic pocket
(A) Structure of the hMob1-phosphopeptide complex. The peptide is shown as yellow sticks
and hMob1 is shown in ribbon representation overlaid with a transparent electrostatic
potential surface. Surface electrostatic potential between −10 kT (red) and +10 kT (blue) is
shown. (B) View of the basic pocket of Mob1. pSer is coordinated by hMob1 K132, R133,
and R136, and the SO42− ion by yeast Mob1 R253, R254, and R257. (C) Binding of
phosphorylated (pT78) or nonphosphorylated (T78) Nud1 peptides to GST-Mob1 or GSTmob1-3RA as in Fig. 3C. (D) Mob1 localization in anaphase A31500 and A31504 cells. (E)
Growth of A2587, A32452, A32586, and A32589 cells on YEPRG and YEPD plates as in
Fig. 1E. (F) Dbf2 kinase activity and cell cycle progression in MOB1 GAL-mob1-77
(A32818) and mob1-3RA GAL-mob1-77 (A32823) cells grown as in Fig. 1A. (G) Model for
Dbf2-Mob1 activation. (H) Growth of A2587, A5588, A32336, A33289, A33551, and
A33285 cells on YEPD+methioinine, CSM-methionine+glucose, or CSM-methionine
+raffinose and galactose (CSMRG) plates as in Fig. 1E. (I) Anaphase kinetics in A1411,
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A33291, A32668, A33549, A33287, and A33283 cells. Cells were arrested in G1 with αfactor in YEPRaff+ methionine medium and released into CSM-methionine+raffinose
medium to induce expression of the Mob1-Nud1 fusion. Galactose was added (arrow) to
induce expression of GAL-TEV1.
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